A micell-mediated phase separation method for preconcentration of ultra-trace quantities of mercury as a prior step to its determination by UV-Vis spectrophotometry has been developed. The method is based on Hg(II)-dithizone complex formation in alkaline aqueous solution, and extraction of this Hg(II)-dithizone secondary complex into the surfactant-rich phase of the non-ionic surfactant octylphenoxypolyethoxyethanol (Triton X-114). This cloud point method of extraction was carried out at 54 ◦C and the surfactant- rich phase containing analyte was dissolved in 250 µL of ethanol with pH 8. The obtained solution was introduced into a micro quartz cell and the analyte was determined by UV-Vis spectrophotometry. Complexation and cloud point extraction of the mercury in alkaline solution markedly facilitate the phase-separation process, lower the temperature of the cloud point, enhances the complex to background absorption ratio and eliminates the interference of Cu2+, Al3+ and Fe3+. Under the optimal extraction and reaction conditions, detection limits, relative standard deviations and linear calibration graphs were, 0.9 ng/mL, lower than 2% and 3.0–250.0 ng/mL respectively. This detection limit is 15 times lower than one obtained by using the same method but at acidic media. The method was applied to the determination of Mercury in soft water and physiology serum samples.